viruses Article A New Multiplex Real-Time RT-PCR for Simultaneous Detection and Differentiation of Avian Bornaviruses Brigitte Sigrist 1, Jessica Geers 2, Sarah Albini 1, Dennis Rubbenstroth 2,3 and Nina Wolfrum 1,* 1 Department of Poultry and Rabbit Diseases, Institute for Food Safety and Hygiene, Vetsuisse Faculty, University of Zurich, CH-8057 Zurich, Switzerland;
[email protected] (B.S.);
[email protected] (S.A.) 2 Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, 17493 Greifswald, Insel Riems, Germany;
[email protected] (J.G.); dennis.rubbenstroth@fli.de (D.R.) 3 Medical Center, Institute of Virology, University of Freiburg, 79104 Freiburg, Germany * Correspondence:
[email protected]; Tel.: +41-44-635-86-36 Abstract: Avian bornaviruses were first described in 2008 as the causative agents of proventricular dilatation disease (PDD) in parrots and their relatives (Psittaciformes). To date, 15 genetically highly diverse avian bornaviruses covering at least five viral species have been discovered in different bird orders. Currently, the primary diagnostic tool is the detection of viral RNA by conventional or real-time RT-PCR (rRT-PCR). One of the drawbacks of this is the usage of either specific assays, allowing the detection of one particular virus, or of assays with a broad detection spectrum, which, however, do not allow for the simultaneous specification of the detected virus. To facilitate the simultaneous detection and specification of avian bornaviruses, a multiplex real-time RT-PCR assay was developed. Whole-genome sequences of various bornaviruses were aligned. Primers were designed to recognize conserved regions within the overlapping X/P gene and probes were selected to detect virus species-specific regions within the target region.